Compositions and methods of using islet neogenesis peptides and analogs thereof

ABSTRACT

The invention provides peptides and analogs of INGAP and HIP peptides. The peptides and analogs can be used in methods for treating various diseases and conditions. Such diseases and conditions can include impaired pancreatic function, treating a metabolic disease, for example, diabetes, both type 1 and type 2 diabetes, islets induction, expansion and proliferation for transplantation, promoting neuroprotection or nerve regeneration, promoting liver regeneration or inhibiting inflammation.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of medicine andpharmaceuticals, and more specifically to peptide therapies for treatingdiabetes and other diseases.

Diabetes mellitus (DM) afflicts over 300 million people worldwide. Thereare two main types of DM: type 1 DM (T1D) and type 2 DM (T2D). T1Dresults from the body's failure to produce insulin, and requires thepatient to administer insulin daily. T2D results from insulinresistance, a condition in which cells fail to use insulin properly.There are many approved non-insulin therapies for T2D. However, there isa large portion of late stage T2D patients requiring insulinadministration due to the loss of β-cell function as the diseaseprogresses.

Development of diabetes is associated with substantial losses inpancreatic islet mass. At the time of diagnosis, over 90% of islet masshas been lost in T1D patients, and approximately 50% has been lost inT2D patients. Many attempts have been made in quest of a potentialstimulus for islet neogenesis, which is considered as the optimaltreatment for both T1D and T2D.

Recently, investigators have shown that islet neogenesis-associatedprotein (INGAP), human prolslet peptide (HIP), glucagon like peptide-1(GLP-1), islet endocrine neuropeptide vasoactive intestinal peptide(VIP), epidermal growth factor and gastrin, and others, are capable ofinducing pancreatic progenitor cells, located in the nonendocrinefraction of the pancreas, to differentiate into fully functional isletsin various animal models. Among these compounds, INGAP peptide(INGAP-PP), a 15-mer peptide derived from the sequence of INGAP at aminoacids 104-118, has been shown to induce islet neogenesis in multipleanimal models, reverse streptozotocin (STZ) induced diabetes in mice,increase C-peptide secretion in T1D patients, and improve glycemiccontrol in T2D patients. Additional biological effects of INGAP-PP havebeen reported, including dose dependent stimulation of expansion ofβ-cell mass and increased insulin secretion and β-cell size. In proof ofconcept human studies, there was an effect with an improvement ofglucose homeostasis, confirmed by HbA1c reduction at 90 days in patientswith T2D, and by a significant increase in C-peptide secretion inpatients with T1D. However, the short plasma half-life of INGAP-PP andthe need for administration in a high dose have significantly limitedclinical applications of this peptide.

HIP, the bioactive peptide encoded by a portion of the human REG3A gene,is the human homolog of the INGAP peptide. Previous studies have shownthat treatment of human pancreatic ductal tissues with HIP stimulatedthe production of insulin. Administration of HIP improved glycemiccontrol and increased islet number in diabetic mice. The stabilized formof HIP is currently being tested in a single ascending dose clinicaltrial with the goal of exploring the tolerability, safety andpharmacokinetics. It is of note that total daily doses of 60, 120, 240,480, and 720 mg are planned. Although there is a lack of data on theefficacious dose of HIP, the planned clinical trial doses infer thepeptide's low potency.

Thus, there exists a need to develop additional drugs for treatment ofdiabetes or other diseases associated with impaired pancreatic function.The present invention satisfies this need, and provides relatedadvantages as well.

SUMMARY OF INVENTION

The invention provides peptides and analogs or INGAP and HIP peptides.The peptides and analogs can be used in methods for treating variousdisease and conditions. Such diseases and conditions can includeimpaired pancreatic function, treating a metabolic disease, for example,diabetes, both type 1 and type 2 diabetes, islets induction, expansionand proliferation for transplantation, promoting neuroprotection ornerve regeneration, promoting liver regeneration or inhibitinginflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the comparison of ARIP cell (a rat pancreatic ductal cellline) proliferation in the presence or 100 nM of INGAP Scrambled PP 1(Peptide 3), INGAP-PP (Peptide 1), and a selected peptide analog,Peptide 7 (see Table 2).

FIG. 2 shows a stability comparison in culture medium of INGAP-PP(Peptide 1) and selected peptide analogs, Peptide 7 and Peptide 8 (seeTable 2).

FIG. 3 shows a stability comparison in mouse plasma of INGAP-PP(Peptide 1) and selected peptide analogs, Peptide 12, Peptide 16 andPeptide 29 (see Table 2).

FIG. 4 shows a stability comparison in human plasma of INGAP-PP(Peptide 1) and selected peptide analogs, Peptide 12 and Peptide 16 (seeTable 2).

FIG. 5 shows a stability comparison in mouse plasma or HIP (Peptide 2)and selected peptide analogs, Peptide 52 and Peptide 54 (see Table 3).

FIGS. 6A-6C show the efficacy comparison of INGAP-PP (Peptide 1), INGAPScrambled PP 1 (Peptide 3) and a selected peptide analog, Peptide 7 (seeTable 2) in STZ induced diabetic mice model. FIG. 6A: Blood glucose (BG)after 21 day treatment; FIG. 6B: Fasting insulin levels after 21 daytreatment; FIG. 6C: Area under curve (AUC) of glucose measured in oralglucose tolerance test (OGTT) after 21 day treatment.

FIG. 7 shows the number of islets defined by area ranges (arbitrarymorphometric units) for equal randomly selected fields (n≥7) for animalstreated with naive and Peptide 3, Peptide 1 or Peptide 7.

FIG. 8 shows the increase of glucose-stimulated insulin secretion ofislets with or without the co-incubation of selected peptides (10μg/mL), Peptide 12, Peptide 16 and Peptide 1 (see Table 2).Co-incubation with 100 nM Glucagon like peptide-1 (GLP-1) was includedas a positive control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds, in particular peptide orpeptide analogs, that exhibit properties useful for treating a varietyof diseases and conditions, particularly diseases and conditionsrelating to diabetes. The peptides and analogs of the invention areadditionally useful for treating impaired pancreatic function, treatinga metabolic disease, ex vivo islet induction, expansion andproliferation For transplantation, increasing the survival oftransplanted islets in vivo, promoting neuroprotection or nerveregeneration, promoting liver regeneration or inhibiting inflammation.

As disclosed herein, the present invention provides a series oilINGAP-PP and HIP analogs with comparable or improved stability andactivities compared to the wild-type peptides (see Tables 2 and 3). Theimproved pharmaceutical properties of these peptide analogs make themparticularly suitable for clinical development. The present inventionalso provides pharmaceutical compositions comprising a compoundaccording to the present invention and the use of compounds according tothe present invention for preparing medicaments for treating metabolicdiseases, including but not limited to type 1 diabetes (T1D) and type 2diabetes (T2D). The invention further provides the compositions of theinvention in suitable formulations, including sustained releaseformulations.

As described previously, a hamster protein was identified that promotedpancreatic islet neogenesis and was termed islet neogenesis associatedprotein (INGAP)(see U.S. Pat. No. 5,834,590). A pentadecapeptidefragment of INGAP, referred to herein as INGAP-PP, has been describedand shown to reverse diabetes in a mouse model (Rosenberg et al., Ann.Surg. 240:875-884 (2004); US publication 2006/0009516; see also USpublication 2008/0171704; Kapur et al., Islets 4:1-9 (2012); Chang etal., Mol. Cell. Endocrinol. 335:104-109 (2011); Borelli et al.,Regulatory Peptides 131:97-102 (2005): Dungan et al., DiabetesMetabolism Res. Rev. 25:558-565 (2009); Zha et al., J. Endocrinol.Invest. 35:634-639 (2012); Wang et al., J. Cell Physiol. 224:501-508(2010); Petropavlovskaia et al., J. Endocrinol. 191:65-81 (2006);Taylor-Fishwick et al., Pancreas 39:64-70 (2010); Rosenberg,Diabetologia 39:256-262 (1996): Madrid et al., Regulatory Peptides157:25-31 (2009); and Taylor-Fishwick et al., J. Endocrinol. 100:729-737(2006)). A human peptide, termed human prolslet peptide (HIP) has alsobeen described (Levetan et al., Endrocrin. Pract. 14:1075-1083 (2008);US publication 2011/0280833). The present invention provides analogs ofINGAP-PP and HIP peptides, including the peptides or analogs of Tables 2and 3 that are not parent INGAP-PP or HIP peptides, that exhibitunexpected and beneficial properties over the INGAP-PP and HIP peptides.

As used herein, the term “peptide” refers to a polymer of two or moreamino acids. The peptide can be modified to include analogs,derivatives, functional mimetics, pseudopeptides, and the like, so longas the peptide comprises a polymer of at least two amino acids. Themeaning of the term “peptide” is well known to those skilled in the art.In general, a peptide includes two or more amino acids joined by anamide bond between the carboxyl group of one amino acid residue and theamino group or the adjacent amino acid residue. As described herein, apeptide can comprise naturally occurring amino acids or non-naturallyoccurring amino acids.

As used herein, the term “analog” refers to a variant or a parentmolecule, for example, a parent peptide. For example, an analog of aparent peptide can include a variant, where one or more amino acids aresubstituted relative to the parent peptide. An analog can also include amodification of a parent peptide, including but not limited to,non-naturally occurring amino acids, D amino acids, modified amino-and/or carboxy-terminal (N- or C-terminal) amino acids, in particularmodifications of the amino group on the N-terminus and/or modificationof the carboxyl group in the C-terminus, fatty acid modifications,peptidomimetics, pseudopeptides, and the like, as disclosed herein.Exemplary modifications are described in more detail below.

As used herein, the phrase “impaired pancreatic function” refers to adisease or condition associated with the pancreas, where the pancreasexhibits a decreased function compared to that of a normal or healthyindividual. Exemplary diseases or conditions associated with impairedpancreatic function include, but are not limited to, type 1 diabetes,type 2 diabetes, latent autoimmune diabetes in adults (LADA), impairedfasting glucose, impaired glucose tolerance, insulin deficiency, fastinghyperinsulinemia, insulin resistance, or impaired fasting insulinlevels, or a combination thereof. Such diseases and conditions arediscussed in more detail below.

As described herein, the invention provides peptide analogs of INGAP-PPand HIP peptides. Table 1 shows the sequence of INGAP-PP and HIPpeptides, as well as various scrambled versions of the peptides that areused as negative controls in experiments described herein or can be usedas negative controls in comparative studies with INGAP-PP, HIP or thepeptides analogs of the invention.

TABLE 1 INGAP-PP and HIP Peptides and Control Scrambled Peptides.Peptide ID for Patent Application / SEQ ID NO Sequence 1H-IGLHDPSHGTLPNGS-OH 2 H-IGLHDPTQGTEPNGE-OH 3 H-SHPNG SGTIG LHDPL-OH 4H-SSTGG GDIPP HLLHN-OH 5 H-DGGTP QPGNW IELTH-OH

As described herein, various analogs or INGAP-PP are provided aspeptides or analogs of the invention. Exemplary INGAP-PP peptide analogsor the invention are provided in Table 2.

TABLE 2 Exemplary INGAP-PP Analogs. Peptide ID for Patent Application /SEQ ID NO Sequence 1 H-IGLHDPSHGTLPNGS-OH 6 H-IGLHAPSHGTLPNGS-OH 7H-IGLHDPSHGTLPAGS-OH 8 H-IGLHAPSHGTLPAGS-OH 9 H-IGLHDPSHGTLPAGSK-OH 10H-IGLHDPSHGTLP(Aib)GS-OH 11 H-IGLHDPSHGTLP(N-methyl-L-Alanine) GS-OH 12Ac-IGLHDPSHGTLPAGS-OH 13 H-(D-Isoleucine)GLHDPSHGTLPAGS-OH 14H-(L-NorValine)GLHDPSHGTLPAGS-OH 15 H-(L-NorLeucine)GLHDPSHGTLPAGS-OH 16Ac-IGLHDPSHGTLPNGS-OH 17 H-(D-Isoleucine)GLHDPSHGTLPNGS-OH 18H-IGLHDPSHGTEPNGS-OH 19 H-IGLHDPSQGTLPNGS-OH 20 H-IGLHDPTHGTLPNGS-OH 21H-IGLHDPSHGTLPNGE-OH 22 H-IGLHDPSHGTLPNGK-OH 23 H-IGLHDPSHGTLPAGK-OH 24H-IGLHDPSHGTEPAGS-OH 25 H-IGLHDPSQGTLPAGS-OH 26 R-IGLHDPTHGTLPAGS-OH 27H-IGLHDPSHGTLPAGE-OH 28 H-IGLHDPSHGTLPAG-NH2 29 Ac-IGLHDPSHGTLPAGS-NH230 Ac-IGLHDPSHGTLPAG-NH2 31 Ac-IGLHDPSHGTLPNGS-NH2 32H-IGLHDPSHGTLPNGS-NH2 33 H-IGLHDPSHGTLPNGSC-OH 34 Ac-IGLHDPSHGTLPNGSC-OH35 H-IGLHDPSHGTLPNGSC-NH2 36 Ac-IGLHDPSHGTLPNGSC-NH2 37H-IGLHDPSHGTLPNGC-OH 38 Ac-IGLHDPSHGTLPNGC-OH 39 H-IGLHDPSHGTLPNGC-NH240 Ac-IGLHDPSHGTLPNGC-NH2 41 H-IGLHDPSHGTLPAGS-NH2 42H-IGLHDPSHGTLPAGSC-OH 43 Ac-IGLHDPSHGTLPAGSC-OH 44H-IGLHDPSHGTLPAGSC-NH2 45 Ac-IGLHDPSHGTLPAGSC-NH2 46H-IGLHDPSHGTLPAGC-OH 47 Ac-IGLHDPSHGTLPAGC-OH 48 H-IGHDPSHGTLPAGC-NH2 49Ac-IGLHDPSHGTLPAGC-NH2 73 IGLHDPSHGTLPAG 74 IGLHDPSHGTLPNG 75Ac-IGLHDPSHGTLPNG 76 IGLHDPSHGTLPNG-NH2 77 Ac-IGLHDPSHGTLPNG-NH2

As described herein, various analogs of HIP are provided as peptides oranalogs of the invention. Exemplary HIP peptide analogs of the inventionare provided in Table 3.

TABLE 3 Exemplary HIP Analogs, Peptide ID for Patent Application /SEQ ID NO Sequence 2 H-IGLHDPTQGTEPNGE-OH 50 H-IGLHDPTQGTEPAGE-OH 51H-IGLHDPTQGTEPAGE-OH 52 Ac-IGLHDPTQGTEPAGE-OH 53H-(D-Isoleucine)GLHDPTQGTEPAGE-OH 54 Ac-IGLHDPTQGTEPNGE-OH 55H-(D-Isoleucine)GLHDPTQGTEPNGE-OH 56 H-IGLHDPTQGTEPNGS-OH 57H-IGLHDPTQGTEPAGS-OH 58 H-IGLHDPTQGTLPNGE-OH 59 H-IGLHDPTQGTLPAGE-OH 60Ac-IGLHDPTQGTEPAG-NH2 61 Ac-IGLHDPTQGTEPNGE-NH2 62Ac-IGLHDPTQGTEPAGE-NH2 63 H-IGLHDPTQGTEPNGE-NH2 64 H-IGLHDPTQGTEPNGC-OH65 Ac-IGLHDPTQGTEPNGC-OH 66 H-IGLHDPTQGTEPNGC-NH2 67Ac-IGLHDPTQGTEPNGC-NH2 68 H-IGLHDPTQGTEPAGE-NH2 69 H-IGLHDPTQGTEPAGC-OH70 Ac-IGLHDPTQGTEPAGC-OH 71 H-IGLHDPTQGTEPAGC-NH2 72Ac-IGLHDPTQGTEPAGC-NH2

The invention provides peptides or analogs thereof that are analogs ofINGAP-PP. In one embodiment, the invention provides a peptide or analogthereof comprising a sequence selected from the group consisting ofIGLHDPSHGTLPAGS (SEQ ID NO:7); and IGLHDPSHGTLPAG (SEQ ID NO:73). Forexample, the peptide or analog can comprise a peptide or analog selectedfrom: IGLHDPSHGTLPAGS (SEQ ID NO:7); IGLHDPSHGTLPAG (SEQ ID NO:73),IGLHDPSHGTLPAGSK (SEQ ID NO:9); IGLHDPSHGTLP(Aib)GS (SEQ ID NO:10);IGLHDPSHGTLP(N-methyl-L-Ala)GS (SEQ ID NO:11); Ac-IGLHDPSHGTLPAGS (SEQID NO:12); (D-Ile)GLHDPSHGTLPAGS (SEQ ID NO:13);(L-NorVal)GLHDPSHGTLPAGS (SEQ ID NO:14); (L-NorLeu)GLHDPSHGTLPAGS (SEQID NO:15); IGLHDPSHGTLPAG-NH2 (SEQ ID NO:28); Ac-IGLHDPSHGTLPAGS-NH2(SEQ ID NO:29); Ac-IGLHDPSHGTLPAG-NH2 (SEQ ID NO:30);IGLHDPSHGTLPAGS-NH2 (SEQ ID NO:41); IGLHDPSHGTLPAGSC (SEQ ID NO:42);Ac-IGLHDPSHGTLPAGSC (SEQ ID NO:43); IGLHDPSHGTLPAGSC-NH2 (SEQ ID NO:44);Ac-IGLHDPSHGTLPAGSC-NH2 (SEQ ID NO:45); IGLHDPSHGTLPAGC (SEQ ID NO:46);Ac-IGLHDPSHGTLPAGC (SEQ ID NO:47); IGLHDPSHGTLPAGC-NH2 (SEQ ID NO:48);and Ac-IGLHDPSHGTLPAGC-NH2 (SEQ ID NO:49).

In a particular embodiment of the invention, the peptide or analogthereof can consist of: IGLHDPSHGTLPAGS (SEQ ID NO:7); IGLHDPSHGTLPAG(SEQ ID NO:73); IGLHDPSHGTLPAGSK (SEQ ID NO.9); IGLHDPSHGTLP(Aib)GS (SEQID NO:10); IGLHDPSHGTLP(N-methyl-L-Ala)GS (SEQ ID NO:11);Ac-IGLHDPSHGTLPAGS (SEQ ID NO:12); (D-Ile)GLHDPSHGTLPAGS (SEQ ID NO:13);(L-NorVal)GLHDPSHGTLPAGS (SEQ ID NO:14); (L-NorLeu)GLHDPSHGTLPAGS (SEQID NO.15). IGLHDPSHGTLPAG-NH2 (SEQ ID NO:28); Ac-IGLHDPSHGTLPAGS-NH2(SEQ ID NO:29); Ac-IGLHDPSHGTLPAG-NH2 (SEQ ID NO:30);IGLHDPSHGTLPAGS-NH2 (SEQ ID NO:41): IGLHDPSHGTLPAGSC (SEQ ID NO:42);Ac-IGLHDPSHGTLPAGSC (SEQ ID NO:43); IGLHDPSHGTLPAGSC-NH2 (SEQ ID NO:44);Ac-IGLHDPSHGTLPAGSC-NH2 (SEQ ID NO:45); IGLHDPSHGTLPAGC (SEQ ID NO:46);Ac-IGLHDPSHGTLPAGC (SEQ ID NO:47): IGLHDPSHGTLPAGC-NH2 (SEQ ID NO:43);or Ac-IGLHDPSHGTLPAGC-NH2 (SEQ ID NO:49).

In another embodiment of the invention, additional INGAP-PP analogs areprovided. An embodiment of the invention provided herein includes apeptide or analog thereof comprising a peptide or analog selected fromthe group consisting of: Ac-IGLHDPSHGTLPNGS (SEQ ID NO.16);(D-Ile)GLHDPSHGTLPNGS (SEQ ID NO:17); Ac-IGLHDPSHGTLPNGS-NH2 (SEQ IDNO:31); IGLHDPSHGTLPNGS-NH2 (SEQ ID NO:32); IGLHDPSHGTLPNGSC (SEQ IDNO:33); Ac-IGLHDPSHGTLPNGSC (SEQ ID NO:34); IGLHDPSHGTLPNGSC-NH2 (SEQ IDNO:35); Ac-IGLHDPSHGTLPNGSC-NH2 (SEQ ID NO:36); IGLHDPSHGTLPNGC (SEQ IDNO:37); Ac-IGLHDPSHGTLPNGC (SEQ ID NO:38); IGLHDPSHGTLPNGC-NH2 (SEQ IDNO:39); Ac-IGLHDPSHGTLPNGC-NH2 (SEQ ID NO:40), IGLHDPSHGTLPNG (SEQ IDNO:74); Ac-IGLHDPSHGTLPNG (75); IGLHDPSHGTLPNG-NH2 (76); andAc-IGLHDPSHGTLPNG-NH2 (77).

In a particular embodiment of the invention, the peptide or analogthereof consists of: Ac-IGLHDPSHGTLPNGS (SEQ ID NO:16);(D-Ile)GLHDPSHGTLPNGS (SEQ ID NO:17); Ac-IGLHDPSHGTLPNGS-NH2 (SEQ IDNO:31); IGLHDPSHGTLPNGS-NH2 (SEQ ID NO:32); IGLHDPSHGTLPNGSC (SEQ IDNO:33); Ac-IGLHDPSHGTLPNGSC (SEQ ID NO:34); IGLHDPSHGTLPNGSC-NH2 (SEQ IDNO:35); Ac-IGLHDPSHGTLPNGSC-NH2 (SEQ ID NO:36); IGLHDPSHGTLPNGC (SEQ IDNO:37); Ac-IGLHDPSHGTLPNGC (SEQ ID NO:38); IGLHDPSHGTLPNGC-NH2 (SEQ IDNO39); Ac-IGLHDPSHGTLPNGC-NH2 (SEQ ID NO:40); IGLHDPSHGTLPNG (SEQ IDNO:74); Ac-IGLHDPSHGTLPNG (75); IGLHDPSHGTLPNG-NH2 (76); orAc-IGLHDPSHGTLPNG-NH2 (77).

Further INGAP-PP peptide analogs are provided herein. In still anotherembodiment, the invention provides a peptide or analog thereofcomprising a sequence selected from the group consisting of:IGLHAPSHGTLPNGS (SEQ ID NO:6); IGLHAPSHGTLPAGS (SEQ ID NO:8);IGLHDPSHGTEPNGS (SEQ ID NO:18); IGLHDPSQGTLPNGS (SEQ ID NO:19);IGLHDPTHGTLPNGS (SEQ ID NO:20); IGLHDPSHGTLPNGE (SEQ ID NO:21);IGLHDPSHGTLPNGK (SEQ ID NO:22); IGLHDPSHGTLPAGK (SEQ ID NO:23);IGLHDPSHGTEPAGS (SEQ ID NO:24); IGLHDPSQGTLPAGS (SEQ ID NO:25); andIGLHDPTHGTLPAGS (SEQ ID NO26); IGLHDPSHGTLPAGE (SEQ ID NO:27).

For example, the invention provides a peptide or analog thereofcomprising a peptide or analog selected from: IGLHAPSHGTLPNGS (SEQ IDNO:6); IGLHAPSHGTLPAGS (SEQ ID NO:8); IGLHDPSHTEPNGS (SEQ ID NO:18);IGLHDPSQGTLPNGS (SEQ ID NO:19); IGLHDPTHGTLPNGS (SEQ ID NO:20);IGLHDPSHGTLPNGE (SEQ ID NO:21); IGLHDPSHGTLPNGK (SEQ ID NO:22);IGLHDPSHGTLPAGK (SEQ ID NO:23); IGLHDPSHGTEPAGS (SEQ ID NO:24);IGLHDPSQGTLPAGS (SEQ ID NO:25); IGLHDPTHGTLPAGS (SEQ ID NO:26); andIGLHDPSHGTLPAGE (SEQ ID NO:27). In another embodiment, the inventionprovides a peptide or analog thereof consisting of: IGLHAPSHGTLPNGS (SEQID NO:6); IGLHAPSHGTLPAGS (SEQ ID NO:8); IGLHDPSHGTEPNGS (SEQ ID NO:18);IGLHDPSQGTLPNGS (SEQ ID NO:19); IGLHDPTHGTLPNGS (SEQ ID NO:20);IGLHDPSHGTLPNGE (SEQ ID NO:21); IGLHDPSHGTLPNGK (SEQ ID NO:22);IGLHDPSHGTLPAGK (SEQ ID NO 23); IGLHDPSHGTEPAGS (SEQ ID NO:24);IGLHDPSQGTLPAGS (SEQ ID NO:25); IGLHDPTHGTLPAGS (SEQ ID NO:26); andIGLHDPSHGTLPAGE (SEQ ID NO:27).

The invention additionally provides HIP analogs. In an embodiment of theinvention, the invention provides a peptide or analog thereof comprisingthe sequence IGLHDPTQGTEPAGE (SEQ ID NO:50). In an embodiment of theinvention, the peptide or analog can comprise a peptide or analogselected from: IGLHDPTQGTEPAGE (SEQ ID NO:50); IGLHDPTQGTEP(Aib)GE (SEQID NO:51); Ac-IGLHDPTQGTEPAGE (SEQ ID NO:52); (D-Ile)GLHDPTQGTEPAGE (SEQID NO:53); Ac-IGLHDPTQGTEPAG-NH2 (SEQ ID NO:60); Ac-IGLHDPTQGTEPAGE-NH2(SEQ ID NO:62); IGLHDPTQGTEPAGE-NH2 (SEQ ID NO:68); IGLHDPTQGTEPAGC (SEQID NO:69); Ac-IGLHDPTQGTEPAGC (SEQ ID NO:70); IGLHDPTQGTEPAGC-NH2 (SEQID NO:71); and Ac-IGLHDPTQGTEPAGC-NH2 (SEQ ID NO:72). In a particularembodiment, the peptide or analog thereof consists of: IGLHDPTQGTEPAGE(SEQ ID NO:50); IGLHDPTQGTEP(Aib)GE (SEQ ID NO:51); Ac-IGLHDPTQGTEPAGE(SEQ ID NO:52); (D-Ile)GLHDPTQGTEPAGE (SEQ ID NO:53); andAc-IGLHDPTQGTEPAG-NH2 (SEQ ID NO60); Ac-IGLHDPTQGTEPAGE-NH2 (SEQ IDNO:62); IGLHDPTQGTEPAGE-NH2 (SEQ ID NO:68); IGLHDPTQGTEPAGC (SEQ IDNO:69); Ac-IGLHDPTQGTEPAGC (SEQ ID NO:70); IGLHDPTQGTEPAGC-NH2 (SEQ IDNO:71); or Ac-IGLHDPTQGTEPAGC-NH2 (SEQ ID NO:72).

In another embodiment, the invention provides additional HIP peptideanalogs. For example, the invention provides a peptide or analog thereofcomprising a peptide or analog selected from the group consisting of:Ac-IGLHDPTQGTEPNGE (SEQ ID NO:54); (D-Ile)GLHDPTQGTEPNGE (SEQ ID NO:55);Ac-IGLHDPTQGTEPNGE-NH2 (SEQ ID NO:61); IGLHDPTQGTEPNGE-NH2 (SEQ IDNO:63); IGLHDPTQGTEPNGC (SEQ ID NO:64); Ac-IGLHDPTQGTEPNGC (SEQ IDNO:65); IGLHDPTQGTEPNGC-NH2 (SEQ ID NO:66); and Ac-IGLHDPTQGTEPNGC-NH2(SEQ ID NO:67). In a particular embodiment, the peptide or analogthereof can consist of Ac-IGLHDPTQGTEPNGE (SEQ ID NO:54);(D-Ile)GLHDPTQGTEPNGE (SEQ ID NO:55); Ac-IGLHDPTQGTEPNGE-NH2 (SEQ IDNO:61); IGLHDPTQGTEPNGE-NH2 (SEQ ID NO:63); IGLHDPTQGTEPNGC (SEQ IDNO:64); Ac-IGLHDPTQGTEPNGC (SEQ ID NO:65); IGLHDPTQGTEPNGC-NH2 (SEQ IDNO:66); or Ac-IGLHDPTQGTEPNGC-NH2 (SEQ ID NO:67).

In another embodiment of the invention, a peptide or analog thereof cancomprise a sequence selected from the group consisting of:IGLHDPTQGTEPNGS (SEQ ID NO:56); IGLHDPTQGTEPAGS (SEQ ID NO:57);IGLHDPTQGTLPNGE (SEQ ID NO:58); and IGLHDPTQGTLPAGE (SEQ ID NO:59). Forexample, the peptide or analog thereof can comprise a peptide or analogselected from: IGLHDPTQGTEPNGS (SEQ ID NO:56); IGLHDPTQGTEPAGS (SEQ IDNO:57); IGLHDPTQGTLPNGE (SEQ ID NO:58); and IGLHDPTQGTLPAGE (SEQ IDNO:59). In a particular embodiment, peptide or analog thereof canconsist of: IGLHDPTQGTEPNGS (SEQ ID NO:56); IGLHDPTQGTEPAGS (SEQ IDNO:57); IGLHDPTQGTLPNGE (SEQ ID NO:58); or IGLHDPTQGTLPAGE (SEQ IDNO:59).

As described herein, the peptides or analogs of the invention includeanalogs of INGAP-PP and HIP that can be peptides having the standard 20naturally occurring amino acids, as well as other naturally and/ornon-naturally occurring amino acids. The peptides as described hereingenerally use conventional nomenclature. For example, some peptides aredesignated H-XXX-OH, and it is understood by those skilled in the artthat these can designate unmodified amino-(H-) or carboxy-(—OH) termini.The amino acid sequence can also be represented without an indication ofa modification on the amino- or carboxy-terminus. It is understood bythose skilled in the art that peptides described herein, unless aspecific modification is indicated on the N- or C-terminus, can includeunmodified and modified amino- and/or carboxy-termini on a peptidecomprising a specified amino acid sequence or peptide analog. Thus, apeptide or analog comprising a designated amino acid sequence caninclude additional amino acids on the N- and/or C-terminus as well asmodified amino acids of the designated sequence. A peptide or analogcomprising a designated peptide or analog similarly can include modifiedamino acids and/or additional amino acids, unless the N- and/orC-terminus comprises a modification that precludes the addition of anamino acid, for example through a peptide bond. Such modifications caninclude, for example, an acetylated N-terminus and/or amidatedC-terminus.

As described herein, the peptides or analogs of the invention cancomprise a modification. It is understood by those skilled in the artthat a number of modifications can be made to a peptide or analog.Exemplary modifications include, but are not limited to, an acetylatedN-terminus, an amidated C-terminus, a D amino acid, a modified aminoacid, a fatty acid modification, or a combination thereof. Any of anumber of well known modifications of a peptide or amino acid can beincluded in a peptide or analog of the invention. For example,derivatives can include chemical modifications of the polypeptide suchas alkylation, acylation, carbamylation, iodination, or any modificationwhich derivatizes the polypeptide. Modifications of a peptide or analogcan include modified amino acids, for example, hydroxyproline orcarboxyglutamate, and can include amino acids that are not linked bypeptide bonds.

It is understood by those skilled in the art that any of a number orwell known methods can be employed to produce peptides or analogs or theinvention (see, for example, Protein Engineering: A practical approach(IRL Press 1992); Bodanszky, Principles of Peptide Synthesis(Springer-Verlag 1984), Lloyd-Williams et al, Tetrahedron 49:11065-11133(1993); Kent, Ann. Rev. Biochem. 57:957-989 (1988); Merrifield, J. Am.Chem. Soc., 85:2149-2154 (1963); Merrifield, Methods Enzymol. 289:3-13(1997)). A particularly useful method to produce peptides or analogs ofthe invention is via chemical synthesis using well known methods ofpeptide synthesis. Chemical synthesis is particularly useful forintroducing non-naturally occurring amino acids, modified amino acidsand/or a modified N- and/or C-terminus. For example, an advantage ofusing chemical synthesis to prepare a peptide or analog of the inventionis that (D)-amino acids can be substituted for (L)-amino acids, ifdesired. The incorporation of one or more (D)-amino acids can confer,for example, additional stability of the peptide in vitro or,particularly, in vivo, since endogenous endoproteases generally areineffective against peptides containing (D)-amino acids. Peptides havingD amino acids can also be designated herein using the well knownnomenclature of a small letter for the corresponding single letter codefor an amino acid.

If desired, the reactive side group of one or more amino acids in apeptide or analog of the invention can be modified or amino acidderivatives can be incorporated into the peptide. Selective modificationof a reactive group of a peptide or analog can impart desirablecharacteristics upon a peptide or analog. The choice of including such amodification is determined, in part, by the characteristics required ofthe peptide. For example, a peptide or analog can have a free carboxylterminus or can be modified so that the C-terminus is amidated (seeTables 2 and 3). Similarly, a peptide or analog can have a free aminoterminus or can be modified so that the N-terminus is acetylated (Tables2 and 3) In addition, the peptides or analogs of the invention canoptionally be amidated on the C-terminus and acetylated on theN-terminus. Other modifications of the N- and/or C-terminus of a peptideor analog can also be included within the meaning of a modification.

Other modifications of a peptide or analog of the invention can include,but are not limited to, 2-Aminoadipic acid (Aad); 3-Aminoadipic acid(bAad); beta-Alanine, beta-Aminopropionic acid (bAla); 2-Aminobutyricacid (Abu); 4-Aminobutyric acid, piperidinic acid (4Abu); 6-Aminocaproicacid (Acp); 2-Aminoheptanoic acid (Ahe); 2-Aminoisobutyric acid (Aib);3-Aminoisobutyric acid (bAib); 2-Aminopimelic acid (Apm); 2,4Diaminobutyric acid (Dbu), Desmosine (Des); 2,2′-Diaminopimelic acid(Dpm); 2,3-Diaminopropionic acid (Dpr); N-Ethylglycine (EtGly);N-Ethylasparagine (EtAsn); Hydroxylysine (Hyl): allo-Hydroxylysine(allyl); 3-Hydroxyproline (3-Hyp); 4-Hydroxyproline (4Hyp); Isodesmosine(Ide); allo-Isoleucine (alle); N-Methylglycine (MeGly; sarcosine);N-Methylisoleucine (Melle); 6-N-Methyllysine (MeLys); N-Methylvaline(MeVal); Norvaline (Nva); Norleucine (Nle), and Ornithine (Orn). It isunderstood that all modified alpha-amino acids can be substituted withthe corresponding beta-, gamma- or omega-amino acids.

Another modification of a peptide or analog or the invention includesfatty acid modification. Thus, a peptide or analog of the invention canbe modified by acylation with aliphatic groups, including C2, C4, C6,C8, C10, C12, C14, C16, C18, C20 or longer chains. The peptide or analogcan also be modified by isoprenylation and/or phosphatidylinositol (PI).Other amino acid, peptide or protein modifications are well known tothose skilled in the art (see, for example, Glazer et al., Chemicalmodification of proteins: Selected methods and analytical procedures,Elsevier Biomedical Press, Amsterdam (1975)).

The invention also includes mimetics of the peptides or analogsdisclosed herein, also referred to as peptidomimetics. Mimeticsencompass chemicals containing chemical moieties that mimic the functionof the peptide. For example, if a peptide contains two charged chemicalmoieties having functional activity, a mimetic places two chargedchemical moieties in a spatial orientation and constrained structure sothat the charged chemical function is maintained in three-dimensionalspace. Thus, a mimetic orients functional groups of a peptide or analogof the invention such that the functional activity of a peptide oranalog is retained.

Mimetics or peptidomimetics can include chemically modified peptides,peptide-like molecules containing nonnaturally occurring amino acids,peptoids and the like, and have the functional activity of the peptideor analog upon which the peptidomimetic is derived (see, for example,Burger's Medicinal Chemistry and Drug Discovery 5th ed., vols. 1 to 3(ed. M. E. Wolff; Wiley Interscience 1995)). Methods for identifying apeptidomimetic are well known in the art and include, for example, thescreening of databases that contain libraries of potentialpeptidomimetics (Allen et al., Acta Crystallogr. Section B, 35:2331(1979)) or using molecular modeling (Rusinko et al., J. Chem. Inf.Comput. Sci. 29 251 (1989)). Mimetics or peptidomimetics can providedesirable properties such as greater stability, for example, whenadministered to a subject, such as during passage through the digestivetract and, therefore, can be useful for oral administration.

A variety of mimetics or peptidomimetics are known in the art including,but not limited to, peptide-like molecules which contain a constrainedamino acid, a non-peptide component that mimics peptide secondarystructure, or an amide bond isostere. A mimetic or peptidomimetic thatcontains a constrained, non-naturally occurring amino acid can include,without limitation, an α-methylated amino acid, α-, α-dialkylglycine orα-aminocycloalkane carboxylic acid; an ^(N)α-^(C)α-cyclized amino acid;an ^(N)α-methylated amino acid, a β- or γ-amino cycloalkane carboxylicacid; an α,β-unsaturated amino acid; a β,β-dimethyl or β-methyl aminoacid; a β-substituted-2,3-methane amino acid, an N-Cδ or ^(C)α-^(C)δcyclized amino acid; a substituted praline or another amino acidmimetic. A mimetic or peptidomimetic which mimics peptide secondarystructure can contain, without limitation, a nonpeptidic β-turn mimic;γ-turn mimic; or mimic of helical structure, each of which is well knownin the art. As non-limiting examples, a peptidomimetic also can be apeptide-like molecule which contains an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylene-sulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and othermimetics or peptidomimetics of a peptide or analog of the invention canbe used.

The invention also provides pseudopeptide derivatives of peptides oranalogs of the invention. Pseudopeptides are known in the art aspeptides in which a peptide bond (amide bond) in a peptide is modifiedto an amide bond surrogate (see, for example, Cudic and Stawikowski,Mun-Rev Organic Chem. 4:268-280 (2007); Anderson, in NeuropeptideProtocols, Brent and Carvell, eds. 73:49-60 (1996)). Exemplary amidebond surrogates include, but are not limited to, peptidosulfonamides,phosphonopeptides, depsides and depsipeptides, oliogureas, azapeptidesand peptoids (see Cudic and Stawikowski, supra, 2007) as well asmethylene amino, thioether and hydroxyethylene derivatives, and the like(Anderson, supra, 1996).

The peptides or analogs of the invention can be produced using methodswell known to those skilled in the art, including chemical synthesis ofthe peptides or analogs using well known methods of peptide synthesis,as described herein. Thus, when the peptides or analogs include one ormore non-standard amino acids, it is more likely that they will beproduced by a chemical synthetic method. In addition to using chemicalsynthesis of peptides or analogs, the peptides or analogs can beproduced by expression from encoding nucleic acids. This is particularlyuseful for peptides or analogs that include only naturally occurringamino acids. In such a case, a nucleic acid encoding the peptidesequence can be prepared using well known methods (see Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999)).Generally such a nucleic acid will be expressed recombinantly in asuitable host organism such as a bacterium, yeast, mammalian or insectcell, and the like. Production in bacteria can be particularly usefulfor large scale production of a peptide or analog of the invention. Thepeptide can be expressed in the organism and purified using well knownpurification techniques.

A nucleic acid molecule encoding the peptide or analog of the inventioncan be cloned into an appropriate vector, particularly an expressionvector, and the encoded peptide or analog can be expressed in a hostcell or using an in vitro transcription/translation reaction, therebyproviding a means to obtain large amounts of the peptide or analog.Optionally, the recombinant peptide can be produced as a fusion with atag, such as a His tag, to facilitate identification and purification.Suitable vectors, host cells, in vitro transcription/translationsystems, and tag sequences are well known in the art and commerciallyavailable.

The peptide or analog can be expressed as a single copy, in apolycistronic expression vector, or optionally can be expressed as asingle open reading frame with multiple copies of the peptide sequence.In such a case, the peptide can be obtained by expressing an openreading frame containing multiple copies of the peptide sequence,resulting in expression of a polypeptide with multiple copies of thepeptide. The polypeptide can be post-translationally processed into apeptide or analog of the invention, for example, by engineeringappropriate proteolytic cleavage sites between the copies of the peptideand cleaving the polypeptide into the peptide or analog of theinvention. Although such a recombinant method will generally be usedwhen the peptide or analog of the invention is a peptide containing onlynaturally occurring amino acids, it is also understood that such amethod can be employed with expression hosts suitably engineered toexpress non-naturally occurring amino acids. Additionally, it isunderstood that a peptide or analog expressed recombinantly canoptionally be chemically modified to introduce a desired amino acidmodification or N- and/or C-terminal modification using well knowchemical modification methods (see Glazer et al., supra, 1975).

Thus, the invention additionally provides nucleic acids encodingpeptides or analogs of the invention. Such nucleic acids include, forexample, nucleic acids encoding any of the amino acid sequences of SEQID NOS:6-73. Thus, when the analogs include only one or moresubstitutions with standard amino acids, the analogs can be expressedfrom an expression vector using well known methods, as disclosed herein.

The peptides or analogs of the invention can comprise a sequence orpeptide or analog as disclosed herein. In the case of a peptide oranalog comprising an amino acid sequence or peptide, the peptide willgenerally have a length of 20 amino acids or less. For example, thepeptide or analog can have a length of 19 amino acids or less, 18 aminoacids or less, 17 amino acids or less. Thus, a peptide or analog of theinvention, as disclosed herein, can have a length of 10 amino acids, 11amino acids, 12 amino acids, 13 amino acids 14 amino acids (see Peptide73, Peptide 74), 15 amino acids, 16 amino acids, 17 amino acids, 18amino acids, 19 amino acids, or 20 amino acids. In the case of shorterpeptides, it is understood by those skilled in the art that the shorterpeptide includes a fragment of a disclosed peptide or analog, forexample, by deletion of one or more amino acids on the N- and/orC-terminus of a disclosed peptide or analog, that retains functionalactivity, including but not limited to one or more of the biologicalactivities of peptides and analogs of the invention, as disclosedherein. Nevertheless, it is understood that a peptide can also compriselonger amino acid lengths, so long as the functional activity of thepeptide or analog is retained. Thus, a peptide or analog can have alength or less than 150 residues, less than 130 residues, less than 120residues, less than 110 residues, less than 100 residues. less than 90residues, less than 80 residues, less than 70 residues, less than 60residues, less than 50 residues, less than 45 residues, less than 40residues, less than 35 residues, less than 30 residues, less than 25residues, less than 24 residues, less than 23 residues, less than 22residues, less than 21 residues, less than 20 residues, less than 19residues, less than 18 residues, or less than 17 residues. It isunderstood by those skilled in the art that, where a peptide or analogof the invention comprises a sequence found within a known longersequence such as a wild type full length protein, the peptide or analogof the invention specifically excludes such a full length sequence.

The invention also provides peptides and analogs of the invention in apharmaceutically acceptable salt form that is well known to thoseskilled in the art. A particularly useful salt form is acetate orhydrochloride salt form. Nevertheless, it is understood by those skilledin the art that any of a number of suitable salt forms are available.When the peptide or analog of the invention contains an acidic or basicmoiety, it can be provided as a pharmaceutically acceptable salt (see,for example. Berge et al., J. Pharm. Sci. 1977, 66, 1-19, and Handbookof Pharmaceutical Salts, Properties, and Use; Stahl and Wermuth, Ed.;Wiley-VCH and VHCA; Zurich, Switzerland, 2002).

Suitable acids for use in the preparation of pharmaceutically acceptablesalts include, but are not limited to, acetic acid, 2,2-dichloroaceticacid, acylated amino acids, adipic acid, alginic acid, ascorbic acid,L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, boric acid, (+)-camphoric acid, camphorsulfonic acid,(+)-(1S)-camphor-10-sulfonic acid, cupric acid, caproic acid, caprylicacid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid,D-glucuronic acid, L-glutamic acid, α-oxoglutaric acid, glycolic acid,hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid,(+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid,maleic acid, (−)-L-malic acid, malonic acid, (±)-DL-mandelic acid,methanesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinicacid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid,pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid,saccharic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaricacid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, andvaleric acid.

Suitable bases for use in the preparation of pharmaceutically acceptablesalts, including, but not limited to, inorganic bases, such as magnesiumhydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, orsodium hydroxide; and organic bases, such as primary, secondary,tertiary, and quaternary, aliphatic and aromatic amines, includingL-arginine, benethamine, benzathine, choline, deanol, diethanolamine,diethylamine, dimethylamine, dipropylamine, diisopropylamine,2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine,isopropylamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine,morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine,piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine,pyridine, quinuclidine, quinoline, isoquinoline, secondary amines,triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine,2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

The invention also provides peptides and analogs of the invention in acomposition. The composition can optionally be formulated with apharmaceutically acceptable carrier to produce a pharmaceuticalcomposition, which can be administered to the individual, which can be ahuman or other mammal. A pharmaceutically acceptable carrier can be, forexample, water, sodium phosphate buffer, phosphate buffered saline,normal saline or Ringer's solution or other physiologically bufferedsaline, or other solvent or vehicle such as a glycol, glycerol, an oilsuch as olive oil or an injectable organic ester

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or increase theabsorption of the peptide or analog of the invention. Suchphysiologically acceptable compounds include, for example, carbohydratessuch as glucose, sucrose or dextrans; antioxidants such as ascorbic acidor glutathione; chelating agents such as ethylenediamine tetraaceticacid (EDTA), which disrupts microbial membranes; divalent metal ionssuch as calcium or magnesium; low molecular weight proteins; or otherstabilizers or excipients. One skilled in the art would know that thechoice of a pharmaceutically acceptable carrier, including aphysiologically acceptable compound, depends, for example, on the routeof administration of the composition. Suitable carriers and theirformulations are well known in the art (see, for example, Remington: TheScience and Practice of Pharmacy, 19th ed., ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. (1995); and Remington's PharmaceuticalSciences, 18th ed., Mack Publishing Company, Easton, Pa. (1990)).Typically, an appropriate amount of a pharmaceutically-acceptable saltis used in the formulation to render the formulation isotonic. The pH ofthe solution is generally from about 5 to about 8, for example, fromabout 7 to about 7.5.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH, as described above. Pharmaceuticalcompositions can include carriers, thickeners, diluents, buffers,preservatives, surface active agents and the like in addition to themolecule of choice such as the peptides or analogs of the invention.Pharmaceutical compositions can also include one or more activeingredients such as antimicrobial agents, anti-inflammatory agents,anesthetics, and the like.

Further carriers include sustained or controlled release preparationssuch as semipermeable matrices of solid hydrophobic polymers covalentlyor non-covalently bound to the peptide or analog, which matrices are inthe form of shaped articles, for example, films, liposomes, non-liposomelipid complex or microparticles, and the like, or other biocompatiblepolymers well known to those skilled in the art (see, for example, U.S.Pat. Nos. 6,824,822 and 8,329,648). Liposomes, which consist ofphospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, BocaRaton, Fla. 1984). Various drug delivery methods are well known to thoseskilled in the art (Langer, Nature 392(Suppl). 5-10 (1998); Langer etal., Nature 428:487-492 (2004)). It will be apparent to those personsskilled in the art that certain carriers can be selected depending upon,for instance, the route of administration and concentration ofcomposition being administered.

The pharmaceutical composition can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. It is understood that a variety of routes ofadministration are useful for the peptides, analogs and methods of theinvention. Such routes encompass systemic and local administration andinclude, without limitation, intravenous injection, intraperitonealinjection, intramuscular injection, subcutaneous injection, transdermaldelivery, transdermal diffusion or electrophoresis, inhalableadministration, oral administration, local injection, intracavity, andextended release delivery devices including locally implanted extendedrelease devices such as bioerodible or reservoir-based implants.Administration can be topically (including ophthalmically, vaginally,rectally, intranasally), orally, by inhalation, or parenterally, forexample by intravenous drip, subcutaneous, intraperitoneal orintramuscular injection.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Insulin is a well known peptide therapeutic, somethods used for delivery of insulin are particularly amenable as adelivery method for peptides or analogs of the invention, including butnot limited to syringes, pens, infusion pumps, inhalers, mouth sprays,pills, and the like.

Guidance on appropriate doses for the peptides or analogs of theinvention is provided in Dungan et al., Diabetes Metab. Res. Rev.,25:558-565 (2009). In particular, human clinical trials with INGAPpeptide provide an indication of suitable possible closer for thepeptides or analogs of the invention. Since the peptides or analogs ofthe invention exhibit improved efficacy over the parent INGAP peptide(see Examples), the peptides or analogs of the invention can beadministered at effective doses that are lower than that used for INGAP.

As described herein, the peptides and analogs of the invention areparticularly useful for treating certain diseases and disorders. Forexample, the peptides or analogs of the invention can be used fortreating impaired pancreatic function, treating a metabolic disease,promoting neuroprotection or nerve regeneration, promoting liverregeneration or inhibiting inflammation. Thus, the inventionadditionally provides compositions of the invention for treatingimpaired pancreatic function, treating a metabolic disease, promotingneuroprotection or nerve regeneration, promoting liver regeneration orinhibiting inflammation. The use of the peptides and analogs of theinvention in such therapeutic applications are described below in moredetail.

If desired, the peptides or analogs of the invention can be administeredin combination. For example, a combination of two or more peptides oranalogs of the invention, including those disclosed herein and shown inTables 2 and 3, can be administered for a method of treatment asdisclosed herein. Such a combination can be administered concurrently,either in separate formulations or combined into the same formulation,depending on the peptides being administered and the compatibility ofthe formulations for the peptides or analogs untie invention.Alternatively, the two or more peptides or analogs of the invention canbe administered sequentially, including on the same day or staggered onseparate days.

Furthermore, it is understood by those skilled in the art that thepeptide and analogs of the invention can optionally be administered withdrugs or therapeutic agents for treating a condition. For example, inthe case of treating diabetes or related conditions, other anti-diabeticdrugs can be administered with the peptides or analogs of the invention.It is understood that such a co-administration can occur concurrently,either in separate formulations or combined into the same formulation,depending on the drugs being administered and the compatibility of theformulations for the peptides or analogs of the invention.Alternatively, the co-administration can occur sequentially, includingon the same day or staggered on separate days. One skilled in the artwill understand appropriate administration regimens suitable foreffective deliver of a peptide or analog of the invention with anotherdrug or therapeutic agent.

In the case of treating insulin or related disorders, suitableanti-diabetic drugs include, but are not limited to, insulin,pramlintide, GLP-1 receptor agonists, oral anti-diabetic agents, and thelike. Exemplary anti-diabetic drugs include, but are not limited to,insulin, meglitinides, for example, repaglinide (Prandin™) andnateglinide (Starlix™), sulfonylureas, for example, glipizide(Glucotrol™), glimepiride (Amaryl™), and glyburide (DiaBeta™, Glynase™);dipeptidy peptidase-4 (DPP-4) inhibitors, for example, saxagliptin(Onglyza™), sitagliptin (Januvia™), and linagliptin (Tradjenta™);biguanides, for example, metformin (Fortamet™, Glucophage™);thiazolidinediones, for example, rosiglitazone (Avandia™) andpioglitazone (Actos™); alpha-glucosidase inhibitors, for example,acarbose (Precose™) and miglitol (Glyset™); amylin mimetics, forexample, pramlintide (Symlin™); and incretin mimetics, for example,exenatide (Byetta™) and liraglutide (Victoza™). Thus, in methods anduses of the invention for treating diabetes or related conditions, ananti-diabetic drug can be administered with a peptide or analog of theinvention.

Type 1 diabetes and latent autoimmune diabetes in adults (LADA) are bothautoimmune diseases. Therefore, in the case of a subject having type 1diabetes or LADA, another therapeutic agent that can be administeredwith a peptide or analog of the invention can be, for example, an immunemodulatory agent. The immunomodulatory agent can be used to block orreduce the destruction of neogenic islet or beta cells associated withautoimmunity. Exemplary immunodulatory agents include, but are notlimited to, sirolimus (rapamycin, Rapamune™), tacrolimus (FK 506,Prograf™), antithymocyte globulin, basiliximab (Simulect™), DiaPep277™,and the like.

As described herein, the peptides and analogs of the invention exhibitunexpected properties over that or the parent INGAP-PP and HIP peptides,including the peptides and analogs of Tables 2 and 3 that are not theparent INGAP-PP or HIP peptides. As disclosed herein, peptides andanalogs of the invention exhibit improved stability in culture mediumand plasma over that of the parent peptide (see Example III). Thepeptide analogs of the invention also were effective at significantlyimproving blood glucose, fasting insulin and oral glucose tolerance (seeExample IV). The peptide analogs of the invention also exhibit asignificantly increased islet neogenic effect than the parent peptide(see Example V). Further, the peptide analogs of the invention exhibit asignificantly increased ability to stimulate insulin secretion inprimary pancreatic islet cells (see Example VI). Additionally, peptideanalogs of the invention exhibited superior pharmacokinetic properties(see Example VII). The numerous unexpected and superior properties ofthe peptides and analogs of the invention indicate that the peptides andanalogs of the invention, including the peptides and analogs at Tables 2and 3 that are not the parent INGAP-PP or HIP peptides, can be utilizedfor therapeutic applications.

In a further embodiment, the invention provides a method forameliorating a sign or symptom associated with impaired pancreaticfunction comprising administering a peptide or analog of the invention.A disease or condition associated with impaired pancreatic functionincludes, but is not limited to, type 1 diabetes, type 2 diabetes,latent autoimmune diabetes in adults (LADA), impaired fasting glucose,impaired glucose tolerance, insulin deficiency, lastinghyperinsulinemia, insulin resistance, or impaired fasting insulinlevels, or a combination thereof. The pancreas produces insulin forregulation of blood glucose. In conditions such as type 1 and type 2diabetes and LADA, the body cannot respond normally to glucoseproduction, leading to a number of related conditions (see CecilTextbook of Medicine, Bennett and Plum, eds., 20th ed., W.B. Saunders,Philadelphia (1996), Harrison's Principles of Internal Medicine, Fauciet al., eds., 14th ed., McGraw-Hill, New York (1998)). It is understoodby those skilled in the art that such conditions, which are correlatedwith decreased function of the pancreas, are included within the meaningof impaired pancreatic function.

Diabetes mellitus is a serious metabolic disease that is defined by thepresence of chronically elevated levels of blood glucose(hyperglycemia). This state of hyperglycemia is the result of a relativeor absolute lack of activity of the peptide hormone, insulin. Insulin isproduced and secreted by the β-cells of the pancreas. Insulin promotesglucose utilization, protein synthesis, and the formation and storage ofcarbohydrate energy as glycogen. Glucose is stored in the body asglycogen, a form of polymerized glucose, which may be convened back intoglucose to meet metabolism requirements. Under normal conditions,insulin is secreted at both a basal rate and at enhanced rates followingglucose stimulation, all to maintain metabolic homeostasis by theconversion or glucose into glycogen.

The term diabetes mellitus encompasses several different hyperglycemicstates. These states include type 1 (insulin-dependent diabetes mellitusor IDDM) and type 2 (non-insulin dependent diabetes mellitus or NIDDM)diabetes. The hyperglycemia present in individuals with type 1 diabetesis associated with deficient, reduced, or nonexistent levels of insulinwhich are insufficient to maintain blood glucose levels within thephysiological range. Treatment or type 1 diabetes involvesadministration of replacement doses or insulin, generally by aparenteral route. The hyperglycemia present in individuals with type 2diabetes is initially associated with normal or elevated levels ofinsulin; however, these individuals are unable to maintain metabolichomeostasis due to a state of insulin resistance in peripheral tissuesand liver and, as the disease advances, due to a progressivedeterioration of the pancreatic β cells which are responsible for thesecretion of insulin. Thus, initial therapy of type 2 diabetes may bebased on diet and lifestyle changes augmented by therapy with oralhypoglycemic agents such as sulfonylureas. Insulin therapy is oftenrequired, however, especially in the latter states of the disease, inorder to produce some control of hyperglycemia and minimizecomplications of the disease.

The invention additionally provides a method for ameliorating a sign orsymptom associated with a metabolic disease in a subject comprisingadministering a peptide or analog of the invention to the subject. Sucha metabolic disease includes, but is not limited to, diabetes,pre-diabetes or metabolic syndrome.

Prediabetes is a condition where blood sugar level is higher than normalbut not yet high enough to be classified as type 2 diabetes. Metabolicsyndrome is a name for a group of risk factors that occur together andincrease the risk for coronary artery disease, stroke, and type 2diabetes. The two most important risk factors for metabolic syndrome areextra weight around the middle and upper parts of the body (centralobesity)(so-called “apple-shaped”) and insulin resistance, where thebody uses insulin less effectively than normal. Insulin is needed tohelp control the amount of sugar in the body. As a result, blood sugarand fat levels rise. Metabolic syndrome is considered to be present if asubject has three or more or the following signs: blood pressure equalto or higher than 130/85 mmHg; fasting blood sugar (glucose) equal to orhigher than 100 mg/dL; large waist circumference (length around thewaist)(men, 40 inches or more; women, 35 inches or more); low HDLcholesterol (men, under 40 mg/dL; women, under 50 mg/dL); triglyceridesequal to or higher than 150 mg/dL.

One skilled in the art will readily understand and can readily determineappropriate indicators of the effectiveness of the peptides or analogsof the invention at ameliorating a sign or symptom associated with acondition or disease associated with impaired pancreatic function and/ormetabolic disease. For example, both type 1 and type 2 diabetes are awell characterized diseases with a number of known parameters fordiagnosing and/or monitoring the progression of the disease and/or tomonitor the effectiveness of a therapy. Such parameters include, but arenot limited to, plasma glucose levels, fasting glucose levels, oralglucose tolerance test (OGTT), insulin levels, fasting insulin levels,glycosylated hemoglobin levels, and the like.

The peptides or analogs of the invention can therefore be used toameliorate any one or more of the signs or symptoms associated withimpaired pancreatic function and/or metabolic disease. In the case ofdiabetes, such signs or symptoms include, but are not limited to,impaired glucose tolerance, increased blood glucose (in particular above200 mg/dl), increased fasting blood glucose (in particular above 140mg/dl), increased postprandial (after eating) blood glucose, insulindeficiency, fasting hyperinsulinemia, insulin resistance, impairedfasting insulin levels, increased glycosylated hemoglobin (HbA1c), andthe like. Such signs or symptoms are well known to those skilled in theart and can be routinely determined by those skilled in the art,including tests available through medical testing laboratories. In anembodiment of the invention, the invention provides a method of reducinga sign or symptom associated with a condition such as diabetes, forexample, a method of reducing impaired glucose tolerance, blood glucose,in particular daily average blood glucose concentration, fasting bloodglucose, postprandial (after eating) blood glucose, insulin deficiency,fasting hyperinsulinemia, insulin resistance, impaired fasting insulinlevels, glycosylated hemoglobin (HbA1c), arginine-stimulated C-peptide,advanced glycation end products (AGE), or a combination thereof, byadministering a peptide or analog of the invention. Methods ofmonitoring the effectiveness of a drug for treating diabetes are wellknown to those skilled in the art (see, for example, Cecil Textbook ofMedicine, supra; Harrison's Principles of Internal Medicine supra,Dungan et al., Diabetes Metabolism Res. Rev. 25:558-565 (2009); U.S.Pat. No. 8,329,648). Thus, the invention provides a method of reducingin a diabetic subject impaired glucose tolerance, blood glucose, fastingblood glucose, postprandial blood glucose, insulin deficiency, fastinghyperinsulinemia, insulin resistance, impaired fasting insulin levels,glycosylated hemoglobin (HbA1c), arginine-stimulated C-peptide, advancedglycation end products (AGE), or a combination thereof, by administeringa peptide or analog of the invention to the subject.

As disclosed herein, the peptides and analogs of the invention wereparticularly effective at stimulating pancreatic islet cell growth andinduction of β-cell clusters (see Example V). Exemplary peptides andanalogs of the invention exhibited improved islet neogenic effect overparent peptide (Example V). Thus, the invention additionally provides amethod for stimulating pancreatic islet cell growth by contacting apancreatic islet cell in vitro with a peptide or analog of theinvention, whereby proliferation of the pancreatic islet cell isstimulated. In another embodiment, the invention provides a method ofproducing a population of pancreatic islet cells, comprising contactingone or more pancreatic islet cells in vitro with a peptide or analog ofthe invention, whereby proliferation of the one or more pancreatic isletcells is stimulated and a population of pancreatic islet cells isproduced. The methods of the invention can be used for ex vivo isletinduction, expansion and proliferation for transplantation and forincreasing the survival of transplanted islets in vivo. Thus, theinvention provides methods of ex vivo islet expansion and proliferationfor transplantation using the peptides or analogs of the invention bycontacting islet cells in vitro, increasing the islet cell numbers andoptionally using the cells for transplantation. The invention alsoprovides a method of increasing the survival of transplanted islets invivo by administering to a subject a peptide or analog of the invention,wherein the subject is the recipient of transplanted islet cells. Thepeptides or analogs of the invention can thus be used to generate cellsfor transplantation using in vitro and ex vivo methods as well as toincrease survival of transplanted islet cells. Such transplanted cellscan be obtained from the in vitro methods using the peptides or analogsor the invention or from traditional transplant sources of islet cellssuch as cadavers.

In a particular embodiment, the one or more pancreatic islet cells canbe obtained from a subject. The population of pancreatic islet cellsproduced by stimulating proliferation of the pancreatic islet cells canbe used, for example, for transplantation into a subject and restorationof pancreatic islet cell function. Thus, a method of the invention canfurther comprise the step of transplanting the population of pancreaticislet cells into a subject. In a particular embodiment, the one or morepancreatic cells are obtained from the subject into which the populationof pancreatic islet cells is to be transplanted. Alternatively, thepancreatic islet cells to be transplanted are obtained from a suitabledonor having a compatible blood type.

Transplantation of pancreatic islets has been described previously (see,for example, Shapiro et al., N. Engl. J. Med. 343:230-238 (2000)).Pancreatic islet cells can be obtained from the subject or,alternatively, from a suitable donor, including islet cells harvestedfrom a cadaver. Generally, the transplant recipient is administeredimmunosuppressive drugs to decrease rejection of the islet cells (see,for example, immunosuppressive drugs described herein). The use ofsuitable immunosuppressive drugs are well known in the field of organ orcell transplantation. Thus, in methods of the invention in whichpancreatic islet cells are stimulated to proliferate in vitro to producea population of pancreatic islet cells, such a population can betransplanted into a subject using well known methods of pancreatic isletcell transplantation. In addition, peptides or analogs of the inventioncan be used to induce differentiation of pancreatic ductal cells intoislet cells, in particular beta-cells (see Yatoh et al., Diabetes 56,1802-1809 (2007)). Thus, the invention further provides a method ofdifferentiating pancreatic ductal cells into islet cells by contacting apancreatic ductal cell with a peptide or analog of the invention. Whenthe method is performed where the pancreatic ductal cell is contacted invitro, a population of differentiated pancreatic ductal cells can begenerated and used for transplantation, as described herein.

The invention further provides a method for increasing the number ofpancreatic islet cells in a subject comprising administering a peptideor analog of the invention to the subject. Such a method of therapeutictreatment using peptides or analogs of the invention can be used toincrease pancreatic islet cells in an individual, without the need toharvest pancreatic cells from the individual or identify a suitabledonor and without the need to put the subject through complextransplantation procedures and the frequently required use ofimmunosuppressive agents if using donor cells not obtained from thepatient.

As described previously, INGAP peptide has been shown to improve nervefunction and enhance nerve regeneration in a diabetic mouse model (Tamet al., FASEB J. 18:1767-1769 (2004)). INGAP peptide was also shown toenhance neurite outgrowth in dorsal root ganglia neurons (Tam et al.,Biochem. Biophys. Res. Communic. 291, 649-654 (2002; Tam et al.,NeuroReport 17:189-193 (2006)). As described herein, the peptides andanalogs of the invention are significantly more active than the INGAPparent peptide and are expected to have a similar but more potentactivity than INGAP. Thus, the invention provides a method for promotingneuroprotection or nerve regeneration by contacting a nerve cell with apeptide or analog the invention, thereby stimulating neuroprotectionand/or nerve regeneration. The contacting with a nerve cell can occur invivo or in vitro. In the case where the nerve cell is contacted in vivo,the peptide or analog of the invention is administered to a subject aswith other therapeutic methods disclosed herein. In the case where thenerve cell is contacted in vitro, the neuroprotected cell can be used inan ex vivo application and the cell administered to the subject. Suchmethods of introducing nerve cells by way of transplantation are wellknown to those skilled in the art (see, for example, Dunnett et al.,Brit. Med. Bulletin 53:757-776 (1997)). Such transplantations have beenperformed to treat neurological conditions such as Parkinson's diseaseand Huntington's disease.

The HIP peptide has been described as accelerating liver regeneration(Lieu et al., Hepatol. 42:618-626 (2005). As described herein, thepeptides and analogs of the invention are significantly more active thanthe HIP parent peptide and are expected to have a similar but morepotent activity than HIP. Thus, the invention also provides a method forpromoting liver regeneration by contacting a liver cell with a peptideor analog of the invention, thereby promoting liver regeneration. Thecontacting with a liver cell can occur in vivo or in vitro. In the casewhere the liver cell is contacted in vivo, the peptide or analog of theinvention is administered to a subject as with other therapeutic methodsdisclosed herein. In the case where the liver cell is contacted invitro, the liver cells can be induced to proliferate, for example, toproduce a population of liver cells. The population of liver cells canbe used in an ex vivo application and the cells administered to thesubject. Methods for transplanting or grafting liver cells onto theliver of a subject are well known to those skilled in the art. Thetransplanted cells can be used to reconstitute injured, or metabolicallydefective, liver tissue. Liver cells can be infused into the portal veinor spleen from where cells migrate to the liver and take up permanenceresidence and perform the normal liver metabolic functions (see, forexample, Khan et al., Cell Transplant. 19:409-418 (2010)).

The HIP protein (also referred to as Pancreatitis-associated protein(PAP)) has been found to exhibit anti-inflammatory activity in vivo andin vitro (Closa et al., World J. Gastroenterol. 13:170-174 (2007)).Therefore, the peptides and analogs of the invention are expected toexhibit anti-inflammatory activity. Therefore, the invention furtherprovides a method for inhibiting inflammation by administering a peptideor analog of the invention.

The invention also provides the use of a peptide or analog of theinvention for preparation of a medicament for treating impairedpancreatic function, treating a metabolic disease, promotingneuroprotection or nerve regeneration, promoting liver regeneration orinhibiting inflammation in a subject.

The invention additionally provides use of a peptide or analog of theinvention for preparation of a medicament for treating impairedpancreatic function, treating a metabolic disease, promotingneuroprotection or nerve regeneration, promoting liver regeneration orinhibiting inflammation in a subject. Such uses can be, for example, tocarry out the methods of the invention disclosed herein.

As described herein, the peptides and analogs of the invention can beused in a variety of methods. Such methods include, but not limited to,treating impaired pancreatic function, treating a metabolic disease,promoting neuroprotection or nerve regeneration, promoting liverregeneration or inhibiting inflammation. In many applications of theinvention for a therapeutic application, the peptides or analogs or theinvention are administered. However, it is understood that analternative mode is to use gene therapy to express a peptide or theinvention by administering a suitable gene therapy vector containing anucleic acid encoding the peptide to a subject. Such gene therapymethods are described below in more detail and are well known to thoseskilled in the art (see, for example, Anderson, Nature 392 (Supp.) 25-30(1998)).

A gene delivery vehicle refers to a molecule that can carry insertedpolynucleotides into a host cell. Examples of gene delivery vehicles areliposomes, micells biocompatible polymers, including natural polymersand synthetic polymers; lipoproteins; polypeptides, polysaccharides,lipopolysaccharides; artificial viral envelopes; metal particles, andbacteria, or viruses, such as baculovirus, adenovirus and retrovirus,bacteriophage, cosmid, plasmid, fungal vectors and other recombinationvehicles typically used in the art which have been described forexpression in a variety of eukaryotic and prokaryotic hosts, and may beused for gene therapy as well as for simple protein expression.

A peptide or analog of the invention can be delivered to a cell ortissue using a gene delivery vehicle. Gene delivery, gene transfer,transducing, and the like as used herein. are terms referring to theintroduction of an exogenous polynucleotide (sometimes referred to as atransgene) into a host cell, irrespective of the method used for theintroduction. Such methods include a variety of well-known techniquessuch as vector-mediated gene transfer (by, e.g., viralinfection/transfection, or various other protein-based or lipid-basedgene delivery complexes) as well as techniques facilitating the deliveryof “naked” polynucleotides (such as electroporation, “gene gun” deliveryand various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide can be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art.

A viral vector refers to a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors, alphavirus vectors and the like. Alphavirus vectors, such asSemliki Forest virus-based vectors and Sindbis virus-based vectors, havealso been developed for use in gene therapy and immunotherapy (seeSchlesinger and Dubensky Curr. Opin. Biotechnol. 5:434-439 (1999) andYing, et al. Nat. Med. 5(7):823-827 (1999)).

In aspects where gene transfer is mediated by a retroviral vector, avector construct refers to the polynucleotide comprising the retroviralgenome or part thereof and a therapeutic gene. As used herein,retroviral mediated gene transfer or retroviral transduction carries thesame meaning and refers to the process by which a gene or nucleic acidsequences are stably transferred into the host cell by virtue of thevirus entering the cell and integrating its genome into the host cellgenome. The virus can enter the host cell via its normal mechanism ofinfection or be modified such that it binds to a different host cellsurface receptor or ligand to enter the cell. As used herein, retroviralvector refers to a viral particle capable of introducing exogenousnucleic acid into a cell through a viral or viral-like entry mechanism.Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof and a transgene. Adenoviruses (Ads) are a relatively wellcharacterized, homogenous group of viruses, including over 50 serotypes(see, for example, WO 95/27071). Ads do not require integration into thehost cell genome. Recombinant Ad derived vectors, particularly thosethat reduce the potential for recombination and generation of wild-typevirus, have also been constructed (see, for example, WO 95/00655 and WO95/11984). Wild-type AAV has high infectivity and specificityintegrating into the host cell's genome (see, for example, Hermonat andMuzyczka, Proc. Natl. Acad. Sci. USA 81, 6466-6470 (1984) and Lebkowskiet al., Mol. Cell. Biol. 8, 3988-3996 (1988)).

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are well known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

Gene delivery vehicles also include DNA/liposome complexes, micelles andtargeted viral protein-DNA complexes. Liposomes that also comprise atargeting antibody or fragment thereof can be used in the methods ofthis invention. To enhance delivery to a cell, the nucleic acid orproteins of this invention can be conjugated to antibodies or bindingfragments thereof which bind cell surface antigens, for example, a cellsurface marker found on pancreatic islet cells.

In yet another embodiment, the invention provides a method ofintroducing a peptide or analog of the invention into a subject bycontacting a cell with a nucleic acid encoding a peptide or analog ofthe invention. The contacting of a cell with the nucleic acid can occurin vitro, for ex vivo applications, or in vivo. Such methods are oftenreferred to as gene therapy methods. When the cell is contacted invitro, the cells expressing the polynucleotide can be administered tothe subject. Such methods permit the expression of a therapeutic proteinor peptide, such as the peptides or analogs of the invention, fortherapeutic applications. Such therapeutic applications can be used fortreating various diseases and conditions, including but not limited totreating impaired pancreatic function, treating a metabolic disease,promoting neuroprotection or nerve regeneration, promoting liverregeneration or inhibiting inflammation, as disclosed herein.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition or the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Production of Peptides and Peptide Analogs

This example describes the production of peptides and peptide analogs.

All the peptides used in the studies were synthesized by solid phasepeptide synthesis using 9-fluorenylmethoxy carbonyl (Fmoc) chemistry. Inbrief, a pre-weighed amount of 2-chlorotrityl chloride resin (1.6mmol/g) was swelled in dichloromethane (DCM). For peptides with anamidated C-terminus, Rink amide resin was used instead of 2-chlorotritylchloride resin. Fmoc-preactivated amino acids were used for the couplingreactions in the presence of hydroxybenzotriazole (Sigma Chemical Co.,St. Louis, Mo., USA) in dimethylformamide (DMF). Excess amino acids wereused throughout the synthesis. Chain elongation reaction was performedfollowed by Fmoc deprotection in 20% piperidine in DMF. When the chainelongation reaction was finished, the Fmoc protecting groups wereremoved from the N terminus of the peptides by 25% piperidine in DMFfollowed by washing with DMF for four times. For peptides with anacetylated N-terminus, before trifluoroacetic acid (TFA) cleavage, asolution at 20% acetic anhydride dissolved in DMF was added at a ratioof 7 mL/g resin, reacted for 30 mins, followed by 4 times washes withDMF and DCM. Following washing for four times with DMF and DCM, theresin was dried under vacuum. Subsequently, the prepared peptides werecleaved from the resin using standard TFA cleavage procedures in TFAwith 5% H₂O followed by multiple ether extractions. All syntheticpeptides were purified to >95% by reverse-phase high-pressure liquidchromatography performed with a liquid chromatograph. Peptides wereanalyzed by mass spectrometry to confirm the identity and purity.

For in vitro and ex-vivo studies, the above prepared peptides weredissolved in double distilled water to make a stock solution, and in thein vivo efficacy study they were reconstituted in sterile normal salineto reach the desired concentration. The final peptide solution wasfiltered through a 0.22 μm membrane to make it sterile.

The peptides and analogs can also be produced using other well knownmethods, including manufacturing the peptides using a method of peptidesynthesis or expressing nucleic acids that code for the desired peptidesor peptide analogs. Thus, when the analogs include one or morenon-standard amino acids, it is more likely that they will be producedby a chemical synthetic method. When the peptides include only one ormore substitutions with standard amino acids, the peptides can beexpressed from an expression vector using well known expression methods.

The particular peptides used in the experiments below can be found inTables 1-3.

EXAMPLE II Stimulatory Effect of Peptides on Cell Proliferation

This example describes the effect of peptides and analogs on pancreaticcell growth.

To measure cell proliferation, a bromodeoxyuridine (BrdU) ELISA assaywas performed. Briefly, ARIP cells (ATCC (American Type CultureCollection), Manassas, Va. USA), a rat pancreatic ductal cell line, werecultured in F-12K medium (Gibco-BRL, Gaithersburg, Md., USA) containing10% fetal bovine serum (FBS; HyClone, Thermo Fisher Scientific Inc.;Waltham, Mass. USA), 100 μg/ml streptomycin and 100 μg/ml penicillin ina cell incubator. ARIP cells were seeded into 96-well culture plates at8000 or 0 (as blank control) cells/well in a volume of 50 μl cellculture medium and incubated overnight for the following experiments. Onthe second day, after replacing the medium with medium without serum, 50μl serum-free cell culture medium containing test peptides at a seriesconcentrations (final concentrations were 10 μM, 5 μM, 1 μM, 500 nM, 100nM, 50 nM, 10 nM and 1 nM) were added to seeded cells. Medium withoutcompound was added to negative control and background control wells. Themedium was replaced at 24 h and 48 h respectively with fresh medium. At69 hours, the medium was supplemented with 10 μl bromodeoxyuridine(BrdU) labeling solution (except the background control wells) from aBrdU cell proliferation ELISA kit (Roche Applied Science; Indianapolis,Ind. USA), and incubated for an additional 3 hours. At 72 hours,labeling medium was removed, and 200 μl/well of FixDenat solution wasadded. After 30 minutes of incubation time. FixDenat solution wasremoved thoroughly and 100 μl/well of anti-BrdU antibody workingsolution was added and incubated at room temperature (RT) for 90minutes. Antibody conjugate was removed and wells were rinsed threetimes with 250 μl/well. Washing solution (1×PBS). After removing washingsolution, 100 μl/well of Substrate solution was added and incubated atRT for 15 min, then 25 μl/well of 1 M H₂SO₄ was added, and the plate wasincubated for about 1 min on the shaker to mix thoroughly. Theabsorbance at 450 nm (reference wavelength 690 nm) on an EnVision™ platereader (Perkin Elmer, Boston, Mass.) within 5 min. after adding the stopsolution was measured.

To test for cell viability, a CellTiter-Glo™ (CTG) assay (Promega,Madison, Wis.) was performed. Briefly, ARIP cells (ATCC, cat# CRL-1674)were cultured in F-12K medium (Gibco-BRL) containing 10% fetal bovineserum (FBS: HyClone), 100 μg/ml streptomycin and 100 μg/ml penicillin ina cell incubator. ARIP cells were seeded into 96-well culture plates at8000 and 0 (as blank control) cells/well in the volume of 50 μl cellculture medium and incubated overnight for the following experiments. Onthe second day, after replacing the medium with medium without serum, 50μl serum-free cell culture medium containing test peptides at a seriesof concentrations (final concentrations were 10 μM, 5 μM, 1 μM, 500 nM,100 nM, 50 nM, 10 nM and 1 nM) were added to seeded cells. Mediumwithout compound was added to negative control and background controlwells. The medium was replaced at 24 h and 48 h respectively with freshmedium. At 72 hours, 25 μl of CellTiter-Glo® reagent was added to eachwell and mixed on an orbital shaker for 2 mins. Luminescence signal wasquantified on an EnVision™ plate reader after a 10 minute incubation atroom temperature.

FIG. 1 shows the comparison of ARIP cell proliferation in the presenceof 100 nM of INGAP Scrambled PP 1 (Peptide 3), INGAP-PP (Peptide 1), andPeptide 7 (peptides shown in Table 2). FIG. 1 shows that there was anincrease in cell number at a peptide concentration of 100 nM. Peptide 7showed a significantly higher percentage increase in cell numbercompared to the INGAP scrambled peptide, a negative control, andINGAP-PP peptide.

EXAMPLE III Peptide Stability Studies

This example describes stability studies of peptides in variousconditions.

To determine the stability of peptides in culture medium, a certainamount of selected peptides was accurately weighed and dissolved indistilled water to 5 mg/mL as a stock solution. The stock solution wasdiluted to 0.25 mg/mL with F-12K medium (Gibco-BRL, Gaithersburg, Md.,USA) as working solution. A volume of 100 μL of each working solutionwas transferred into individual sample vials. The sample vials wereincubated in a 37° C. incubator for 0, 24, 48 and 72 hours before beinganalyzed and quantitated by HPLC.

FIG. 2 shows the stability of compounds in culture medium. Inparticular, FIG. 2 shows a stability comparison in culture medium ofINGAP-PP (Peptide 1) and selected peptide analogs, Peptide 7 and Peptide3 (see Table 2). As shown in FIG. 2, peptide analogs Peptide 7 andPeptide 8 were significantly more stable than INGAP-PP peptide inculture medium.

The stability of peptides was also tested in mouse and human plasma.Briefly, a certain amount of peptides and eucatropine powder (positivecontrol) was accurately weighed. Test compounds were dissolved in 50%methanol-water solution and diluted to 20 mg/mL, and eucatropine wasdissolved in dimethylsulfoxide (DMSO) and diluted to 10 mM, as a stocksolution. Eucatropine stock solution was diluted to 0.2 mM with DMSO asa working solution. A stop reagent was prepared containing 200 ng/mLmidazolam and tolbutamide in acetonitrile. A volume of 300 μL of stopsolution was added to each well of a 96-well deep-well plate placed onice beforehand.

For the stability studies, peptides and eucatropine were spiked intoplasma respectively, mixed well, and then 100 μL of each mixturesolution was transferred into the pre-cooled stop reagent as 0 timepoint sample. The remaining mixtures were incubated in a 37° C. waterbath with shaking at 100 rpm (n=2). The final incubation concentrationwas 1 μM for eucatropine and 100 μg/mL for all test compounds.

At desired time points, 100 μl, of incubation mixture was transferred tothe stop reagent to precipitate proteins. Samples were vortexed andcentrifuged at RCF 5000-g for 10 minutes, and supernatant wastransferred to a test plate. The samples were analyzed by LC-MS/MS.

Slope was calculated by plotting the natural logarithm of the percentageor remaining amount of test compounds and time, and T₁₂ was calculatedin accordance with the following formula.

$T_{1/2} = \frac{0.693}{- {slope}}$

For stability in mouse plasma, the incubation time was 0, 5, 15, 30 and60 min for Peptide 1 and eucatropine, 0, 15, 30, 60, 120, 240 and 480min for Peptide 12, Peptide 16 and Peptide 29. FIG. 3 shows stability ofcompounds in mouse plasma. In particular, FIG. 3 shows a stabilitycomparison in mouse plasma of INGAP-PP (Peptide 1) and selected peptideanalogs, Peptide 12, Peptide 16 and Peptide 29 (see Table 2). As shownin FIG. 3, peptide analogs Peptide 12, Peptide 16 and Peptide 29exhibited good stability in mouse plasma and were more stable thanINGAP-PP (Peptide 1).

In another stability study in mouse plasma, the incubation time was 0,30, 60 and 120 min for Peptide 2 and eucatropine; 0, 30, 60, 120, 240and 960 min for Peptide 52 and Peptide 54. FIG. 5 shows stability ofcompounds in mouse plasma. In particular, FIG. 5 shows a stabilitycomparison in mouse plasma of HIP (Peptide 2) and selected peptideanalogs, Peptide 52 and Peptide 54 (see Table 3). As shown in FIG. 5,peptide analogs Peptide 52 and Peptide 54 exhibited good stability inmouse plasma and were significantly more stable than HIP (Peptide 2).

For stability in human plasma, the incubation time was 0, 30, 60 and 120min for Peptide 1, Peptide 12, Peptide 16 and eucatropine. FIG. 4 showsthe stability of compounds in human plasma. In particular, FIG. 4 showsa stability comparison in human plasma of INGAP-PP (Peptide 1) andselected peptide analogs, Peptide 12 and Peptide 16 (see Table 2). Asshown in FIG. 4, peptide analogs Peptide 12 and Peptide 16 exhibitedgood stability in human plasma and were significantly more stable thanINGAP-PP (Peptide 1).

These results demonstrate that various peptide analogs exhibit goodstability under various conditions, including culture medium and mouseand human plasma, and exhibit superior stability over INGAP-PP and HIPpeptides.

EXAMPLE IV Efficacy of Peptide Analogs in a Diabetic Mouse Model

This example describes an in vivo efficacy study using a streptozotocin(STZ) induced diabetic mice model.

After acclimatization in the animal facility for one week, 6-8 weeks oldC57BL/6J mice were administered low dose STZ at 40 mg/kg in citratebuffer for 5 consecutive days to establish a TID animal model. Mice withblood glucose greater than 16.7 mmol/L at 5 days post last STZ injectionwere included in the study. These mice were then treated with INGAP-PP(Peptide 1) or Peptide 7 at the closes of 5 mg/kg (2.5 mg/kg, bid (twicea day)) or 25 mg/kg (12.5 mg/kg, bid) for 20 days before sacrifices. Twoadditional groups of diabetic mice were administered either saline or apeptide (Peptide 3) composed of a scrambled sequence of amino acids fromPeptide 1 as control groups. Blood glucose and insulin levels weremeasured, and 20 days post the last dosing of test agents, an oralglucose tolerance test (OGTT) was performed in 6 hour fasted animals todetermine the effect of Peptide 1 and Peptide 7. Blood samples obtainedfrom the tail cut for glucose determination were detected with anACCU-CHEK™ glucometer (Roche, ACCU-CHEK® Active), and insulin levelswere determined with Rat/Mouse Insulin Elisa kit (Millipore, Billerica,Mass. USA). For the OGTT, after the measurement of the basal glucoseconcentration (T=−30 min), mice received an oral glucose challenge at 2g/kg and glucose values were determined by glucometer at 0, 15, 30, 60,90 and 120 min.

FIG. 6 shows the efficacy comparison of INGAP-PP (Peptide 1), INGAPScrambled PP 1 (Peptide 3) and Peptide 7 in STZ induced diabetic micemodel. FIG. 6A shows the blood glucose (BG, mM) on day 21 of treatment.FIG. 6B shows the fasting insulin levels (ng/ml) on day 21 of treatment.FIG. 6C shows the area under curve (AUC) of glucose (T_(0˜120min))measured in an oral glucose tolerance test (OGTT) on day 21 oftreatment.

Administration of Peptide 1 and Peptide 7 (either 5 mg/kg or 25 mg/kg)for 20 days did not affect body weight or pancreas weight. Significantdifferences in blood glucose levels were demonstrated between the mousegroup administered Peptide 7 and the saline control group (FIG. 6A).Moreover, one of the most striking results was that plasma insulinlevels of the Peptide 7 treated animals (25 mg/kg dose group) at the endof the 20-day period were significantly different from saline controlsand almost restored to the level of the naive group (FIG. 6B). Inaddition, the Peptide 7 treated groups also demonstrated improvedglucose tolerance (FIG. 6C).

These results demonstrate that a representative peptide analog, peptide7, was effective at ameliorating signs and symptoms of diabetes in adiabetic mouse model.

EXAMPLE V The Effect of Peptides on Induction of Small β-Cell Clusters

This example describes the effects of peptides on the induction of smallβ-cell clusters in normal C57BL/6J mice.

After a 1-week acclimation, C57BL/6J female mice were randomly dividedinto 4 groups. The two control groups received either 10 mL/kg sterilenormal saline (n=4) or scrambled peptide (Peptide 3, 25 mg/kg) (n=5) viasubcutaneous injection for 10 days. The other two groups receivedINGAP-PP (Peptide 1) or INGAP-PP analog Peptide 7 at a dose of 25 mg/kgper day respectively (n=7 per group) for the same period. Body weightand 6 hour fasting blood glucose were measured before treatment andafter the last dosing of treatment. Plasma and pancreatic insulin werealso measured at the end of the study. On day 11, the pancreas wasremoved from each animal, cleared of fat and lymph nodes, weighed, andfixed in 10% neutral buffered formalin (NBF) for no longer than 24 hoursbefore processing for morphometric analysis.

Compared to the saline group, administration of Peptide 3, Peptide 1, orPeptide 7 to normal mice for 10 days did not affect body weight, bloodglucose, plasma insulin, pancreas insulin, or pancreas weight.Immunohistochemistry analysis was used to determine pancreatic isletsize distribution. FIG. 7 shows pancreatic islet size distribution infemale C57BL/6J mice at 10 days of peptide treatment. For the islet size(expressed as Log [μm²]) ranging from 4.9 to 2.3, there was nodifference for each group, whereas for the islet size ranging from 2.1to 0.7, the numbers increased significantly in the mice treated withPeptide 7 (p<0.05 or 0.01 versus the naive/control group)(FIG. 7). Theincrease in the Peptide 1 treated mice was only observed in islet sizeof 2.1 (p>0.05 versus the naive/control group).

These results indicated the improved islet neogenic effect of designedINGAP-PP analogs. It is of note that among all parameters measured,there was no difference for mice treated with normal saline or scrambledpeptide.

EXAMPLE VI The Effect of Peptides on Glucose-Stimulated InsulinSecretion

This example describes the effect of peptides on glucose-stimulatedinsulin secretion (GSIS).

The pancreases were procured from male adult Sprague-Dawley (SD) rats.After 7 days acclimation, the animals were sacrificed by cervicaldislocation and the entire pancreas was removed and digested withcollagenase to isolate islets. After digestion, islets were maintainedat 37° C. in RPMI 1640 (Carlsbad, Calif., USA) pH 7.4, containing 10%(v/v) fetal calf serum, 1% penicillin/streptomycin, and 10 mM glucose ina humid atmosphere (5% CO₂/95% O₂), without the addition of any compound(control), or with the addition of 100 nM glucagon like peptide-1(GLP-1); or 10 μg/mL Peptide 1, Peptide 12, or Peptide 16, as summarizedin Table 4 below.

TABLE 4 Parameters for Various Groups Tested for Glucose- stimulatedInsulin Secretion (GSIS) 1 2 3 4 5 6 A 1.5 mM 12 mM 12 mM 12 mM 12 mM 12mM Glucose Glucose Glucose; Glucose; Glucose; Glucose; 100 nM 10 μg/ml1.0 μg/ml 10 μg/ml GLP-1 Peptide 1 Peptide 12 Peptide 16 B 1.5 mM 12 mM12 mM 12 mM 12 mM 12 mM Glucose Glucose Glucose; Glucose; Glucose;Glucose; 100 nM 10 μg/ml 10 μg/ml 10 μg/ml GLP-1 Peptide 1 Peptide 12Peptide 16 C 1.5 mM 12 mM 12 mM 12 mM 12 mN 12 mM Glucose GlucoseGlucose; Glucose; Glucose; Glucose; 100 nM 10 μg/ml 10 μg/ml 10 μg/mlGLP-1 Peptide 1 Peptide 12 Peptide 16 D 1.5 mM 12 mM 12 mM 12 mM 12 mM12 mM Glucose Glucose Glucose; Glucose; Glucose; Glucose; 100 nM 10μg/ml 10 μg/ml 10 μg/ml GLP-1 Peptide 1 Peptide 12 Peptide 16

Cultured islets were rinsed in Krebs-Ringer bicarbonate buffer (KRB), pH7.4, previously gassed with a mixture CO₂/O₂ (5/95%), and pre-incubatedin 1.0 ml KRB containing 0.5% (w/v) BSA and 1.5 mM glucose at 37° C. for45 min. After this period, groups of 5 islets were incubated in 0.6 mlKRB with the addition of 1.5 or 12.0 mM glucose, with or without theaddition of peptides for 60 min. At the end of the incubation period,aliquots of the medium were collected for insulin quantitation.

The results of the insulin quantitation are shown in FIG. 8. FIG. 8shows the increase of glucose-stimulated insulin secretion of isletswith or without the co-incubation of selected peptides (10 μg/mL),Peptide 12, Peptide 16 and Peptide 1. Co-incubation with 100 nM Glucagonlike peptide-1 (GLP-1) was included as a positive control. At 12.0 mMglucose concentration, pancreatic islets cultured with peptides GLP-1,Peptide 12 and Peptide 16 released significantly more insulin than thosecultured without the addition of peptides. In particular, INGAP-PPanalogs Peptide 12 and Peptide 16 showed 2-3 fold higher stimulation ofinsulin secretion than GLP-1. In contrast, no stimulation was observedwith the addition of INGAP-PP (Peptide 1) (FIG. 8).

These results demonstrate that INGAP-PP analogs stimulated insulinsecretion from pancreatic islet cells.

EXAMPLE VII Pharmacokinetic Properties of Peptides in Rat and Mouse

This example describes in vivo pharmacokinetic (PK) properties ofpeptides in rat and mouse.

After 7 days acclimation, male Sprague-Dawley (SD) rats weighing 210-250g, or male C57BL/6 mice, weighing 19-24 g, in good health were used inthe study. Peptide 1, Peptide 12 and Peptide 16 were dissolved insterile normal saline and then they were injected via subcutaneous (sc)bolus or intravenous (iv) bolus at the dose level of 25 mg/kg. Threeanimals in each group were used for blood collection at the time pointof 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h post-dose.Blood samples (approximately 400 μL) were collected and placed intotubes containing EDTA-K2 and centrifuged at 8000 rpm for 6 minutes at 4°C. to separate plasma front the samples. The resulting plasma was storedfrozen at −80° C. until being analyzed.

Plasma concentrations of peptides were determined using tandem massspectrometry (LC-MS/MS) analysis. A non-compartmental module ofWinNonlin® Professional 5.2 (Pharsight; St. Louis, Mo.), was used tocalculate PK parameters. Selected PK parameters are presented in Table 5below. The abbreviation AUC_((0-t)) represents area under the curvefront the time of dosing to the time of the last observation, theAUC_((0-∞)) represents area under the curve from the time of dosing toinfinity, and the C_(max) represents maximum concentration detected.

TABLE 5 Pharmacokinetic Parameters in Treated Mice and Rats Study MOUSE(SC) RAT (SC) RAT (IV) Parameters AUC_((0-t)) AUC_((0-∞)) C_(max)AUC_((0-t)) AUC_((0-∞)) C_(max) AUC_((0-t)) AUC_((0-∞)) C_(max) μg/L*hrμg/L*hr μg/L μg/L*hr μg/L*hr μg/L μg/L*hr μg/L*hr μg/L Peptide 1 54.558.3 140.9 298.5 305.1 1238.0 21.9 27.1 48.7 Peptide 12 5873.1 5888.211600.7 8423.3 8480.0 14024.2 12632.5 12633.8 44312.7 Peptide 16 11350.611354.4 14376.4 12127.1 12202.7 18907.7 12191.6 12192.7 45194.9

Compared to INGAP-PP (Peptide 1), the peptide analogs Peptide 12 andPeptide 16 showed marked improved PK properties evidenced by thesignificant increase in the area under the plasma concentration-timecurves (AUC) and the maximum concentration (Cmax) in mouse and rat.

These results demonstrate that the INGAP-PP peptide analogs exhibitedsignificantly improved pharmacokinetic properties over INGAP-PP.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1. A peptide or analog thereof comprising a sequence selected from thegroup consisting of: (SEQ ID NO: 7) IGLHDPSHGTLPAGS; [[and]](SEQ ID NO: 73) IGLHDPSHGTLPAG; (SEQ ID NO: 50) IGLHDPTQGTEPAGE;(SEQ ID NO: 16) Ac-IGLHDPSHGTLPNGS; (SEQ ID NO: 17)(D-Ile)GLHDPSHGTLPNGS; (SEQ ID NO: 31) Ac-IGLHD PSHGT LPNGS-NH2;(SEQ ID NO: 32) IGLHDPSHGTLPNGS-NH2; (SEQ ID NO: 33) IGLHDPSHGTLPNGSC;(SEQ ID NO: 34) Ac-IGLHDPSHGTLPNGSC; (SEQ ID NO: 35)IGLHDPSHGTLPNGSC-NH2; (SEQ ID NO: 36) Ac-IGLHDPSHGTLPNGSC-NH2;(SEQ ID NO: 37) IGLHDPSHGTLPNGC; (SEQ ID NO: 38) Ac-IGLHDPSHGTLPNGC;(SEQ ID NO: 39) IGLHDPSHGTLPNGC-NH2; (SEQ ID NO: 40)Ac-IGLHDPSHGTLPNGC-NH2; (SEQ ID NO: 74) IGLHDPSHGTLPNG;Ac-IGLHDPSHGTLPNG (75); IGLHDPSHGTLPNG-NH2 (76);Ac-IGLHDPSHGTLPNG-NH2 (77); (SEQ ID NO: 54) Ac-IGLHDPTQGTEPNGE;(SEQ ID NO: 55) (D-Ile)GLHDPTQGTEPNGE; (SEQ ID NO: 61)Ac-IGLHDPTQGTEPNGE-NH2; (SEQ ID NO: 63) IGLHDPTQGTEPNGE-NH2;(SEQ ID NO: 64) IGLHDPTQGTEPNGC; (SEQ ID NO: 65) Ac-IGLHDPTQGTEPNGC;(SEQ ID NO: 66) IGLHDPTQGTEPNGC-NH2; (SEQ ID NO: 67)Ac-IGLHDPTQGTEPNGC-NH2; (SEQ ID NO: 6) IGLHAPSHGTLPNGS; (SEQ ID NO: 8)IGLHAPSHGTLPAGS; (SEQ ID NO: 18) IGLHDPSHGTEPNGS; (SEQ ID NO: 19)IGLHDPSQGTLPNGS; (SEQ ID NO: 20) IGLHDPTHGTLPNGS; (SEQ ID NO: 21)IGLHDPSHGTLPNGE; (SEQ ID NO: 22) IGLHDPSHGTLPNGK; (SEQ ID NO: 23)IGLHDPSHGTLPAGK; (SEQ ID NO: 24) IGLHDPSHGTEPAGS; (SEQ ID NO: 25)IGLHDPSQGTLPAGS; (SEQ ID NO: 26) IGLHDPTHGTLPAGS; (SEQ ID NO: 27)IGLHDPSHGTLPAGE; (SEQ ID NO: 56) IGLHDPTQGTEPNGS; (SEQ ID NO: 57)IGLHDPTQGTEPAGS; (SEQ ID NO: 58) IGLHDPTQGTLPNGE; and (SEQ ID NO: 59)IGLHDPTQGTLPAGE.

2-13. (canceled)
 14. The peptide or analog of claim 1, wherein thepeptide or analog thereof comprises a modification.
 15. The peptide oranalog of claim 14, wherein the modification is selected from anacetylated N-terminus, an amidated C-terminus, a D amino acid, amodified amino acid, a fatty acid modification, or a combinationthereof.
 16. The peptide or analog of claim 1, wherein the peptide oranalog thereof has a length of 20 amino acids or less.
 17. A compositioncomprising the peptide or analog of claim
 1. 18. The composition ofclaim 17, further comprising a pharmaceutically acceptable carrier. 19.(canceled)
 20. A method for ameliorating a sign or symptom associatedwith impaired pancreatic function comprising administering the peptideor analog of claim
 1. 21. The method of claim 20, wherein the impairedpancreatic function is type 1 diabetes, type 2 diabetes, latentautoimmune diabetes in adults (LADA), impaired fasting glucose, impairedglucose tolerance, insulin deficiency, fasting hyperinsulinemia, insulinresistance, or impaired fasting insulin level, or a combination thereof.22. (canceled)
 23. A method for stimulating pancreatic islet cellgrowth, comprising contacting a pancreatic islet cell in vitro with thepeptide or analog of claim 1, whereby proliferation of the pancreaticislet cell is stimulated.
 24. A method of producing a population ofpancreatic islet cells, comprising contacting one or more pancreaticislet cells in vitro with the peptide or analog of claim 1, wherebyproliferation of the one or more pancreatic islet cells are stimulatedand a population of pancreatic islet cells is produced. 25-27.(canceled)
 28. A method for increasing the number of pancreatic isletcells in a subject comprising administering the peptide or analog ofclaim
 1. 29. A method for ameliorating a sign or symptom associated witha metabolic disease in a subject comprising administering the peptide ofclaim
 1. 30. The method of claim 29, wherein the metabolic disease isdiabetes, pre-diabetes or metabolic syndrome.
 31. A method of reducingin a diabetic subject impaired glucose tolerance, blood glucose, fastingblood glucose, postprandial blood glucose, insulin deficiency, fastinghyperinsulinemia, insulin resistance, impaired fasting insulin levels,glycosylated hemoglobin (HbA1c), arginine-stimulated C-peptide (AUC), ora combination thereof, by administering a peptide or analog of claim 1to the subject.
 32. A method for promoting neuroprotection or nerveregeneration, comprising contacting a nerve cell with the peptide oranalog of claim
 1. 33. A method for promoting liver regeneration,comprising contacting a liver cell with the peptide or analog ofclaim
 1. 34-35. (canceled)
 36. A method for inhibiting inflammation,comprising administering the peptide or analog of claim
 1. 37-39.(canceled)